Myogenic Control Mechanisms of Circular Muscle of the Rectosigmoid Colon

描述（由申请人提供）：膜微域中信号蛋白的易位、磷酸化和关联强烈表明 PKC1 和 RhoA 在结肠运动中的个体重要作用。 HSP27 的磷酸化对于结肠圆形平滑肌细胞 (CSMC) 收缩过程中 PKC1 和 RhoA 的易位至关重要。 CSMC 的收缩与 HSP27 磷酸化相关，而 S16 处的 HSP20 磷酸化会抑制收缩。成人 CSMC 的初步结果显示：1）脂筏膜组分中存在 cav-1； 2) 乙酰胆碱 (Ach) 诱导的 PKC1、磷酸化 PKC1 (S657) 和 HSP27 向脂筏中的隔离以及 HSP20 从脂筏中的易位增加； 3) PKC1 和 HSP27 与 CSMC 颗粒部分中的小窝蛋白 1 (cav-1) 的关联增加； 4) 沉默 PKC1（PKC1 的 siRNA）对用 PKC1 的 siRNA 处理的人结肠 CSMC 生物工程化的 3 维环（3DBR）中 Ach 诱导的力产生的初始上升和持续阶段均具有抑制作用，表明 PKC1 在力的产生及其维持中发挥作用。来自老年大鼠 CSMC 的初步数据表明，分离的脂筏组分中 cav-1 被耗尽，因此磷酸化 PKC1 (S657) 被耗尽，同时 PKC1 和 HSP27 与 cav-1 的关联性降低。此外，用 DN cav-1 cDNA 转染的成年 CSMC 通过表现出 PKC1 和 HSP27 与 cav-1 的关联减少来模拟老年 CSMC 的 Ach 反应。这与老年大鼠 CSMC 中 Ach 诱导的 3DBR 产生力减少相关。这些数据表明，脂筏、cav-1 和 HSP27 在正常收缩反应以及与衰老 CSMC 相关的小窝形成减少中发挥着至关重要的作用。减少小窝形成可能是受衰老影响的关键因素，也是假定的治疗目标。初步数据表明，wt-cav-1 的异位表达恢复了老化结肠 3DBR 中 Ach 诱导的力产生。因此，我们建议使用多级功能方法来研究收缩信号通路的复杂性。我们将使用活细胞成像、生化和分子生物学工具研究不同蛋白质的时空重组和重新定位，并使用由成年、老化和稳定转染的平滑肌细胞生物工程设计的 3 维环对收缩反应进行实时生理监测。通过这些方法获得的数据将使我们能够：1）辨别负责理解结肠运动生理学的复杂分子机制； 2) 了解并确定受衰老影响的假定破坏机制，这些机制导致结肠收缩迟缓和病理生理学； 3) 确定并测试可能的假定目标，以纠正与年龄相关的病理生理学和结肠蠕动迟缓。 公共健康相关性：总之，我们将利用我们开发的生化、分子和结构生理学工具来检测由于衰老而导致的正常生理运动功能的破坏，并最终设计出治疗方法来纠正这些缺陷。

DESCRIPTION (provided by applicant): Translocation, phosphorylation and association of signaling proteins in membrane microdomains, strongly suggest individual imperative roles of PKC1 and RhoA in colonic motility. Phosphorylation of HSP27 is essential for translocation of PKC1 and RhoA during contraction of colonic circular smooth muscle cells (CSMC). Contraction of CSMC is associated with HSP27 phosphorylation, while HSP20 phosphorylation at S16 inhibits contraction. Preliminary results from adult CSMC show: 1) the presence of cav-1 in lipid raft membrane fractions; 2) increased acetylcholine (Ach)-induced sequestration of PKC1, phospho-PKC1 (S657) and HSP27 into the lipid rafts and translocation of HSP20 out of the lipid rafts; 3) increased association of PKC1 and HSP27 with caveolin 1 (cav-1) in the particulate fraction of CSMC; and 4) silencing of PKC1 (siRNA for PKC1) had an inhibitory effect of both the initial rise and the sustained phase of Ach-induced force generation in 3-dimensional rings bioengineered (3DBR) from human colon CSMC treated with siRNA for PKC1 suggesting a role for PKC1 in force generation and its maintenance. Preliminary data from aged rat CSMC show that isolated lipid raft fractions were depleted of cav-1 and consequently of phospho-PKC1 (S657) concomitant with decreased association of PKC1 and HSP27 with cav-1. Further, adult CSMC transfected with DN cav-1 cDNA, mimicked Ach response of aged CSMC by exhibiting reduced association of PKC1 and HSP27 with cav-1. This correlated with reduced Ach-induced force generation of 3DBR from CSMC of aged rats. These data suggest a crucial role for lipid rafts, cav-1, and HSP27 in normal contractile responses and a reduction in caveolae formation associated with aging CSMC. Reduced caveolae formation could be a critical factor affected by aging and a putative therapeutic target. Preliminary data indicates that ectopic expression of wt-cav-1 reinstated Ach-induced force generation in 3DBR from aged colon. Therefore, we propose to use multilevel functional approaches to study the intricacies of contractile signaling pathways. We will study the spatiotemporal reorganization and relocation of different proteins using live cell imaging, biochemical and molecular biology tools and real time physiological monitoring of contractile response using 3-dimensional rings bioengineered from adult, aged, and stably transfected smooth muscle cells. The data obtained through these approaches will allow us to: 1) Discern the intricate molecular mechanisms responsible for the understanding of the physiology of colonic motility; 2) Understand and identify putative disrupted mechanisms affected by aging that contribute to the sluggishness and pathophysiology of contraction of the colon; and 3) Identify and test the possible putative targets to rectify age-related pathophysiology and sluggishness of colonic motility. Public Health Relevance: In summary, we will utilize biochemical, molecular and structural physiological tools we have developed to detect the disruption of normal physiological motor function due to aging and ultimately design therapies to rectify these defects.